Cruciat Et Al, 1999; De Lonlay Et Al, 2001)
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A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Dissecting the genotype to phenotype relationships of Genomic Disorders Lesley Ruth Hart Submitted to the University of Sussex for the degree of Doctor of Philosophy (DPhil) June 2013 i Declaration I hereby declare that this thesis has not been, and will not be, submitted in whole or in part to another University for the award of any other degree. Signature………………………………………………………………… Date………………….. ii This thesis is dedicated in loving memory of my parents, Andrew and Karen This is for you. “A good name is more desirable than great riches; to be esteemed is better than silver or gold” iii Acknowledgements First and foremost I offer my sincerest gratitude to my supervisor, Professor Mark O’Driscoll. Over the last four years Mark has instilled in me, by example, a strong sense of discipline and integrity and I consider it a true honour to have been able to complete my research in his lab. His continued encouragement, enthusiasm and guidance has made this thesis possible. One simply could not wish for a better or friendlier supervisor. I would also like to thank Professor Antony Carr for his continued support throughout my studies, particularly during the second year of my research. In my daily work, I have been blessed with the most cheerful and fun-loving lab group I could have asked for; Rita Colnaghi, Emily Outwin, Diana Alcantara, Iga Abramowicz and Gill Carpenter. I am immensely grateful for both the support and the happy memories they have given me over the past four years. Much of what I know now is thanks to them and for that, I am eternally thankful. I acknowledge the Medical Research Council for their financial support enabling me to carry out this research. I would also like to express my utmost appreciation for being given the opportunity to carry out my research within the Genome Centre and I thank all the staff for their never-ending assistance and support. I owe my deepest gratitude to my family, partner and friends for supporting and encouraging me to pursue my dreams. In particular my Dad, without whom University would not have been an option and who will forever continue to be my inspiration in life. To my extended family; “The Murray’s” and “The Bourke’s”, your overwhelming support and love has helped me through several hurdles in life. Without that, I could not have gone so far. Thank you. iv University of Sussex Lesley Ruth Hart DPhil Biochemistry Dissecting the genotype to phenotype relationships of Genomic Disorders SUMMARY Over the last decade, major advances in the development and application of microarray-based comparative genomic hybridisation (aCGH) technology have significantly contributed to our understanding of Genomic Disorders. My aims here were to provide insight into the genotype to phenotype relationships of three Genomic Disorders; CUL4B-deleted X-Linked Mental Retardation (XLMR), Wolf-Hirschhorn Syndrome (WHS) and 16p11.2 Copy Number Variant Disorder. CUL4B encodes a structural component of the Cullin-RING-ligase 4-containing class of E3 ubiquitin ligases. CUL4B-deleted XLMR represents a syndromal form of mental retardation whereby patients exhibit other clinical features aside from the MR, such as seizures, growth retardation and disrupted sexual development. I used CUL4B-deleted patient-derived cell lines to investigate the impacts of CUL4B loss on mitochondrial function. I have shown that loss of CUL4B is associated with a distinct set of mitochondrial phenotypes, identifying CUL4B-deleted XLMR as a disorder associated with mitochondrial dysfunction. Furthermore, I have uncovered a reciprocal relationship between CUL4B and Cereblon, providing evidence of a potential role for the CUL4-CRBN E3 ligase complex in maintaining mitochondrial function. Deletion or duplication of the 16p11.2 region is associated with macro-/microcephaly respectively. Here, I have evaluated the cellular consequences of 16p11.2 CNV, specifically with regards KCTD13 expression, DNA replication and checkpoint activation. WHS is typically caused by a small hemizygous telomeric deletion of the 4p16.1 region. Haploinsufficiency of 4p16.1 is associated with microcephaly, growth retardation and complex developmental abnormalities. I investigated the impacts of LETM1 copy number change in WHS patient-derived cells. Here, I have shown that copy number change of LETM1 specifically segregates with mitochondrial dysfunction, likely underlying the seizure phenotype exhibited by the large subgroup of WHS patients whose deletions incorporate LETM1 as well as the rarer instances of the reciprocal duplication. In this thesis I use patient-derived cell lines from three Genomic Disorders as a fundamental tool providing new pathomechanistic insight into the clinical presentation of these conditions. v Table of Contents Chapter One – Introduction: Copy Number Variation and Genomic Disease 1 1.1: Introduction 2 1.2: Copy Number Variation 2 1.3: Recurrent and Non-recurrent CNV formation 5 1.3.1: Recurrent CNVs: Low Copy Repeats and Non-allelic Homologous Recombination 5 1.3.2: Non-recurrent CNVs and Non-Homologous End Joining 12 1.4: Complex Chromosomal Rearrangements 14 1.4.1: Fork Stalling and Template Switching 14 1.4.2: Microhomology-Mediated Break-Induced Repair 15 1.4.3: The Break-Fusion-Bridge Cycle 16 1.5: CNVs, genome stability and cancer 20 1.6: Summary 21 Chapter Two - Materials and Methods 22 2.1: Cell Culture 23 2.2: Antibodies 23 2.3: Reagents 25 2.4: siRNA knockdowns 25 2.5: Extract Preparation 26 2.5.1: Urea-based whole cell extracts 26 2.5.2: Soluble whole cell extracts 26 2.5.3: Mitochondrial extracts 26 2.6: Immunoprecipitation 27 2.7: 5-fluorouridine incorporation and indirect immunofluorescence 27 2.8: Agarose-formamide gel electrophoresis 28 vi 2.9: Semi-Quantitative Duplex PCR 28 2.10: Mitochondrial function; MitoTrackers 29 2.11: Mitochondrial membrane potential; JC-1 29 2.12: Complex I activity 30 2.13: MitoProbe Transition Pore Assay 30 2.14: Calcium measurements 31 2.15: LETM1 overexpression 31 2.16: LETM1 complementation 31 2.17: S-Phase progression 31 2.18: Recovery after HU treatment 32 Chapter Three Results I - X-Linked Mental Retardation and CUL4B; A pathomechanistic dissection of the impact of CUL4B deletion in humans 33 3.1: Introduction 34 3.1.1: The ubiquitin-proteasome pathway of protein degradation 36 3.1.1.1: The role of ubiquitin in the DNA damage response 37 3.1.2: The Cullin family of E3 ligases 42 3.1.2.1: Mouse models of CRL4 deficiency 46 3.1.3: CUL4B X-Linked Mental Retardation 48 3.1.4: CUL4A and CUL4B are functionally distinct; identification of CUL4B-specific substrates 49 3.1.4.1: CUL4B and steroid sex hormone signalling 53 3.1.4.2: CUL4B and WDR5 54 3.1.4.3: CUL4B and Peroxiredoxin III 55 3.1.4.4: A role for CRL4’s in the mTOR signalling pathway 56 3.1.5: CUL4B-dependant Topoisomerase I degradation 58 3.1.6: Mitochondria are dynamic organelles 62 3.1.6.1: Mitochondrial disorders; disorders associated with disrupted mtDNA replication 63 3.1.6.2: Mitochondrial Topoisomerase; Top1mt 68 vii 3.1.7: Summary 68 3.2: Results 70 3.2.1: Characterisation of defective CUL4B function in lymphoblastoid cells from an individual carrying a novel X- chromosome deletion 70 3.2.2: Understanding the impacts of CUL4B deficiency on mitochondrial functioning 72 3.2.2.1: Mitochondrial topoisomerase 1; Top1mt 72 3.2.2.2: Mitotracker Green FM as an indicator of mitochondrial mass 73 3.2.2.3: CUL4B-deleted LBLs exhibit disruption of the mitochondrial membrane potential, Ѱm 77 3.2.2.3.1: JC-1 and Ѱm 79 3.2.2.3.2: Mitotracker Red and Ѱm 80 3.2.2.4: CUL4B-deleted LBLs exhibit reduced ATP levels consistent with ETC dysfunction 87 3.2.2.5: Reactive Oxygen Species; the good and the bad 89 3.2.2.5.1: Mitochondria-derived ROS 89 3.2.2.5.2: MitoSOX: CUL4B LBLs show increased levels of mito-specific ROS 94 3.2.2.6: ETC Complex I activity is unchanged in CUL4B-deleted LBLs 95 3.2.2.7: Intracellular calcium levels are increased in CUL4B- deleted LBLs 99 3.2.2.8: CUL4B-deleted LBLs exhibit hypersensitivity of the mitochondrial permeability transition pore 101 3.2.3: CUL4B is present within mitochondrial extracts 106 3.2.4: siRNA-mediated knockdown of cul4b confirms the mitochondrial phenotypes observed in patient cells 109 3.2.5: Autophagy 113 3.2.5.1: Mitochondria-specific autophagy; mitophagy 114 viii 3.2.5.2: CUL4B-deleted LBLs exhibit disrupted autophagic flux 117 3.2.5.3: Parkin ubiquitination is altered in the context of CUL4B loss 120 3.2.6: Summary 121 3.3: Discussion 124 Chapter Four Results II - Characterizing the reciprocal relationship between CUL4B and Cereblon 128 4.1: Introduction 129 4.1.1: A gene for non-syndromal MR maps to Chromosome 3p25pter 129 4.1.2: Potential roles for CRBN 133 4.1.2.1: CRBN and CNS development; a putative role in learning and memory 133 4.1.2.2: CRBN; a novel thalidomide binding protein 134 4.1.2.3: A putative role for CRBN in the antioxidant response